Breast cancer is the most common malignancy among western women. Although adjuvant and molecularly targeted therapies significantly improve patient survival, breast cancer metastasis remains incurable and is the main cause of morbidity and cancer death in women. Recent genome-wide analysis of human breast cancer identified EphA2 receptor tyrosine kinase as a promising target. We and others found that EphA2 is overexpressed in ~60% of human breast cancer specimens, including drug-resistant HER2 positive tumors and triple-negative/basal-like breast cancer, correlating with poor survival. Ablation of EphA2 in mice inhibited metastatic progression in the HER-dependent MMTV-Neu mammary tumor model. In contrast, expression of ephrin-A1, the prototypic ligand for EphA2, is frequently lost in lymph node metastasis patients and high levels of ephrin-A1 correlates with better survival in patients with metastatic disease, suggesting that ligand-dependent signaling inhibits EphA2-dependent tumor progression and metastatic spread. Therefore, we hypothesize that ephrin-A1 acts as a molecular switch, such that loss of ephrin converts EphA2 functions from tumor suppression to tumor promotion. We are poised to test this model in our newly generated ephrin-A1-null mouse model. In this proposal, we will test the inhibitory role of ephrin-A1 ligand in mammary tumor growth and metastasis in mice and its value as a biomarker for metastatic breast cancer, with the ultimate goal of developing ephrin-A1 as a new prognostic marker for predicting the relative risk of human breast cancer metastasis. We will also determine if EphA2 is required for tumor progression and metastasis induced by loss of ephrin-A1, using both a genetic approach and a selective EphA2 kinase inhibitor (Aim 1). As a first step in investigating the mechanism through which ephrin-A1 inhibits breast cancer progression, we discovered elevated fatty acid synthase (FASN) expression and lipid content, as well as increased glutaminolysis in ephrin-A1-null/MMTV-Neu tumors. Since lipid and glutamine metabolism contributes to breast cancer pathogenesis, we will determine if molecular regulation of lipogenesis and glutaminolysis by ephrin/Eph signaling contributes to growth and metastasis in vivo (Aim 2). Success of this project will not only elucidate the molecular mechanisms that control the switch between tumor promotion versus tumor suppression by EphA2 RTK, but also validate novel EphA2-targeting selective kinase inhibitors for treatment of breast cancer subtypes that are refractory to current therapies, such as triple-negative/basal-like breast cancer or HER2 positive cancers that developed resistance to anti-HER2 agents. In addition, this proposal addresses several areas emphasized in the NCI's provocative questions in cancer biology, including oncogene addiction and tumor metabolism/obesity's influences on cancer.